Nanobody-based VSR7 tracing shows clathrin-dependent TGN to Golgi recycling

Receptor-mediated transport of soluble proteins is nature’s key to empowering eukaryotic cells to access a plethora of macromolecules, either by direct accumulation or as products from resulting biochemical pathways. The transport efficiency of these mechanisms results from the receptor’s capability to capture, transport, and release ligands on the one hand and the cycling ability that allows for performing multiple rounds of ligand transport on the other. However, the plant VACUOLAR SORTING RECEPTOR (VSR) protein family is diverse, and their ligand-specificity and bidirectional trafficking routes and transport mechanisms remain highly controversial. Here we employ nanobody-epitope interaction-based molecular tools to assess the function of the VSR 7 in vivo. We demonstrate the specificity of the VSR7 for sequence-specific vacuolar sorting signals, and we trace its anterograde transport and retrograde recycling route. VSR7 localizes at the cis-Golgi apparatus at steady state conditions and transports ligands downstream to release them in the trans-Golgi network/early endosome (TGN/EE) before undergoing clathrin-dependent recycling from the TGN/EE back to the cis-Golgi.

Also, it is difficult to locate the line for a line plot.If a magnified image is to be included, it would be better to take it from the presented image.The experiment in Fig. 4 is interesting, but the control experiment is lacking, and from the current results, it cannot be determined where the binding took place.For example, authors should show that RFP does not go to the TGN, or GFP does not come to the cis-Golgi, using the cis-Golgi resident SYP3-RFP-NbG.minor I think the reference to the numbers of VSRs and RMRs in page 2 refers to Arabidopsis and does not apply to higher plants in general.I don't understand the rationale for choosing the genes authors used for the phylogenetic analysis in Suppl 1. Also, because tobacco cells are used in this experiment, it would be better to show that there are Class III VSRs in tobacco as well.For VSR5/6, which also belong to Class III, it has been reported that OX of VSR5 in the vsr1vsr4 double mutant promotes AALP secretion (Lee et al., 2013 https://doi.org/10.1104/pp.112.210914).Whether VSR5/6 and VSR7 have different functions in the same Class III or it is due to distinct experimental systems should be verified by additional experiments, if possible.Bottom of page 3, RFP-AFVY is not accumulated in the ER, so it should not be surprising that FRET does not occur.As for the TGN localization of VSR7, VSR7 has been detected in proteome analyses of the TGN using VHAa1 and SYP61 (Groen et al., 2014https://doi.org/10.1021/pr4008464, Drakakaki et al., 2011doi:10.1038/cr.2011.129).These should be cited and discussed in the manuscript.There is no data to exclude the possibility that VSR7 is recycled from the MVB, therefore the title should be an overstatement.More careful discussion would be needed.In the Discussion, the authors state that VSR4 and VSR7 should have the same velocity of anterograde transport in the Golgi.What is the rationale for this?Reviewer #3: Remarks to the Author: In this manuscript Shao et al investigates the trafficking routes of VSR7, an understudied vacuolar sorting receptor and suggest that VSR7 bind cargoes in the cis-Golgi to then release them in downstream TGN/EE compartment.In the paper they use a previously published strategy based on the interaction between GFP and a GFP nanobody to test the trafficking of VSR7.The sensor is able to detect VSR7 in the cis-Golgi.They use FLIM to test interaction of different vacuolar cargoes and show that VSR7 is responsible for the trafficking of an Aleu-RFP cargo.They localize the full length receptor to the cis-Golgi and develop an assay to show that VSR7 recycles back to the Golgi from TGN/EE.Additionally, they show that VSR7 trafficking is clathrin dependent by using a clathrin mutant.
In conclusion they suggest intra Golgi transport of a sub-set cargoes in plants may be a receptordependent process.This is a very interesting model and a novel concept across species as intra-Golgi transport in mammals (debated) and in yeast is thought to be driven by cisternal maturation.In my opinion this is a very bold claim which in this case is unfortunately not supported by the data.It would need careful evaluation and quantification of the imaging data before it can be published.I cannot suggest this work and I feel like I am unable to evaluate this work until proper quantification is carried out.
The authors must carry out quantification and statistics on independent experiments.With crops with one single dot structure, it is impossible to evaluate how sound the data is… For example, in Figure 2B I do see colocalization between ManI and Aleu which makes me worry that the authors may have biasedly picked crops.
I understand that the author claim that cargo binds to the receptor in the cis-Golgi based on their FLIM interaction data (of which I am not an expert and cannot evaluate the soundness…).However, I fail to see data in support of release of cargo in the TGN/EE.I also recommend that the authors move away from using non-color blind friendly red and green to show their microscopy images.

Reviewer #1
The mechanisms and trafficking routes of plant vacuolar sor ng receptor (VSR) proteins are incompletely understood.This manuscript focuses on the ligand specificity and trafficking route of the VSR7 receptor, using nanobodies and epitope tags as molecular markers.Some of the approaches are sophis cated and novel.The basic conclusions are: (a) VSR7 binds ligands in the cis-Golgi but cannot bind ligands in the trans-Golgi network/early endosome (TGN/EE), presumably due to the lower pH, and (b) VSR7 resides at steady-state in the cis-Golgi but travels to the TGN/EE and then recycles in a clathrin-dependent manner.The data presented in support of these conclusions are generally persuasive.In vivo experiments offer strong evidence that VSR7 binds to the vacuolar reporter Aleu-RFP but not to the vacuolar reporter RFP-AFVY, indica ng that VSR7 has specificity for a subset of vacuolar sor ng signals.
Further experiments indicate that the VSR7-ligand interac on can occur in the ER and cis-Golgi but not in the TGN/EE.The analysis of the role of clathrin is a bit convoluted, so those conclusions are more provisional, but clathrin likely has a role in VSR7 trafficking.Overall, this study is technically impressive and it makes a noteworthy contribu on to the field.

Response:
We thank the reviewer for the very posi ve feedback regarding the scien fic significance of this work and the apprecia on of the quality of our work.
My major concern is the interpreta on of the pathway followed by VSR7.The authors propose that VSR7 binds ligands in the cis-Golgi and then releases them in the TGN/EE, but the ra onale for this pathway is puzzling if the Golgi operates by cisternal matura on.Why would a receptor be needed to carry proteins forward to the TGN/EE when matura on will do the job?A relevant review by Robinson and Neuhaus (h ps://pubmed.ncbi.nlm.nih.gov/27262127/)should be cited, because it puts forth a model in which vacuolar sor ng receptors segregate cargoes into specialized domains during transport through the Golgi.To this reader, the new data on VSR7 fit that model nicely.The authors should offer their perspec ve to ensure that their results contribute to our understanding of how VSR7 contributes to vacuolar protein sor ng.

Response:
Here, the reviewer raises an exci ng topic, and we strongly agree with the reviewer's view.Our data regarding the receptor-ligand interac ons and receptor trafficking of the VSRs VSR7 and VSR4 point to a sor ng mechanism of the VSRs, that differs from the classical concept of receptor-mediated transport of soluble ligands in which ligand transport is assumed to occur via vesicle shu les that connect spa ally dis nct compartments, with one providing binding condi ons, while the other provides release condi ons.Regarding VSR-mediated transport in plants, it seems as if the VSRmediated transport of ligands does not occur between spa ally dis nct but temporarily dis nct compartments, with the target compartment emerging as the result of a matura on-based process.In this concept, it seems as if the sor ng func on of a VSR is to bind the ligand at neutral pH in the cis-Golgi cisternae and immobilize it throughout the cisternae's matura on into a VHA-a1-acidified Golgi-independent TGN with EE func onality to prevent its secretory loss.This view is, indeed, in full agreement with the proposed concept from Robinson and Neuhaus, 2016, J. Exp. Bot. 67, 4435-4449.We modified the discussion regarding the VSR-based transport mechanism and added this cita on to the manuscript.

Response:
We apologize for the confusion; we have corrected the mistake.
2) p. 7: "did not colocalize with the TGN/MVB marker" should presumably read "did not colocalize with the TGN/EE marker".

Response:
We apologize for the confusion; we have corrected the mistake.
3) There is evidence for clathrin adaptor-dependent recycling from the TGN to the cis/medial Golgi in yeast and mammalian cells (h ps://pubmed.ncbi.nlm.nih.gov/35429729/).That pathway may be related to the proposed recycling pathway for VSR7.

Response:
We appreciate this informa on and we have included this aspect in the discussion.

Reviewer #2
The authors used transient assay in tobacco protoplasts and the NB-based sensor and showed that VSR7 binds to ALEU in the early secretory pathway and dissociates from it at the TGN/EE.They also showed that VSR7 localizes to the cis-Golgi in a steady state.Using a system that can trap the Nbsensor at the TGN/EE, the authors also showed that VSR7 is recycled a er reaching the TGN.The fact that VSR7 accumulates in the TGN upon overexpression of ARF1DN or Hub indicated that CCV is required for recycling.The trap experiment presented in Fig. 4 is interes ng, but the overall data is not well presented and the conclusions are not convincing.

Response:
We are sorry if the presenta on of the data has caused confusion.In this revised manuscript, we have substan ally revised all Figures according to the sugges ons below.

Major
The problem is that there is no quan ta ve data for colocaliza on/non-colocaliza on.

Response:
We agree with the reviewer, and we have therefore included the quan fica on of the colocaliza on of the respec ve receptors and marker proteins for the analyses of receptor localiza on and receptor trafficking that are presented in Fig. 3,Fig. 4,and Fig. 5 and Supplementary. Fig. 8 by calcula ng the linear Pearson's correla on coefficient (r P ) and nonlinear Spearman's rank correla on coefficient (r S ) of fluorescent signals using the so ware ImageJ with the PSC colocaliza on plugin.We furthermore performed a sta s cal analysis of the respec ve PSCs for a be er apprecia on in general and the localiza on changes observed in the trafficking analysis, in par cular.We present the sca er plots together with the sta s cal analysis as the three new supplementary figures 5, 6, and 7 and added the details to the Methods sec on.In this regard, Supplementary.Fig. 5 confirms that GFP-VSR7 colocalizes with the cis-Golgi marker Man1-RFP but not with the TGN/EE-localizing VSR4, the TGN/EE marker SYP61-RFP, the MVB/LE marker RFP-BP80, or the trans-Golgi marker ST-RFP; Supplementary.Fig. 6 confirms that the endocytosed dual-epitope linker secGFP-SYN, the labeling agent of the trafficking analysis, does not reach the cis-Golgi on its own and also underlines the labeling efficiency of the nanobody-epitope interac on-triggered pos ransla onal labeling of the Nb G -RFP-VSR7 and the SYP61-CFP-Nb S ; Supplementary.Fig. 7 confirms that compared to control cells where the RFP-VSR7 colocalizes with the cis-Golgi marker Man1-CFP but not with the TGN/EE-localizing GFP-VSR4 or the TGN/EE marker SYP61-CFP, the expression of the ARF1 mutant causes a significant reduc on of the colocaliza on between the RFP-VSR7 and the cis-Golgi marker Man1-CFP and a significant increase of the colocaliza on with the GFP-VSR4.Likewise, the coexpression of the GFP-Hub causes a significant rise in the colocaliza on of the RFP-VSR7 with the TGN/EE marker SYP61-CFP.Prolonged expression of the GFP-Hub for 48 hours causes the RFP-VSR7 to colocalize with the MVB/LE marker CFP-BP80, while RFP-VSR4 colocalizes with the TGN/EE marker SYP61-CFP, but not with the MVB/LE marker CFP-BP80.
The lower right corners of pictures in Fig. 2-5 and supplemental data appear to be enlarged images, but where in the image is enlarged is unclear.Many of the dots in the enlarged images are not found in the whole cell image, perhaps because they are taken from another cell or rotated.Also, it is difficult to locate the line for a line plot.If a magnified image is to be included, it would be be er to take it from the presented image.

Response:
We fully agree with the view regarding the insets and the line scan region (LSR) indica ons.For the previous version of the figures, we first imaged the respec ve cell and then took a second image of a sec on of that cell at a higher magnifica on, which we presented as an inset to visualize the respec ve pa ern of the fluorescent signals.During the me this took, the intracellular compartments moved.Therefore, it was not possible to obtain iden cal signal pa erns in both images, and we could not indicate the respec ve regions in the image of the cell.In this revised version, we simplified the situa on by using an enlarged region of the respec ve cell as an inset and framed this region in the image of the respec ve cell with a white square.We also performed the line intensity analysis of a region in the inset and presented this region as an addi onal enlargement, labeled line scan region, LSR, in the respec ve panels.These revisions have been done to Fig. 2, 3, 4, and 5 and Supplementary Fig. 3, 4, and 8.
The experiment in Fig. 4 is interes ng, but the control experiment is lacking, and from the current results, it cannot be determined where the binding took place.For example, authors should show that RFP does not go to the TGN, or GFP does not come to the cis-Golgi, using the cis-Golgi resident SYP3-RFP-NbG.

Response:
We thank the reviewer for apprecia ng the experimental strategy to iden fy TGN/EE transit and recycling of the cis-Golgi localizing VSR7.The reviewer has requested an addi onal control to rule out that the in Fig. 4 observed nanobody-epitope interac on-triggered pos ransla onal labeling of the at steady-state in the cis-Golgi-localizing Nb G -RFP-VSR7 with the endocytosed secGFP-SYN occurred due to TGN/EE to cis-Golgi upstream trafficking of the labeling agent, rather than downstream trafficking of the Nb G -RFP-VSR7 to the TGN/EE and its subsequent recycling to the cis-Golgi, as claimed.For this, the reviewer suggested exposing the Nb G with a red fluorescent cis-Golgi-localizing protein for trapping endocytosed secGFP-SYN molecules upon arrival in the cis-Golgi.We appreciated this sugges on, but for the sake of consistency, we decided to keep using cis-Golgi-localizing marker/anchor constructs based on Man1 throughout the manuscript rather than introducing a new SYP3-based cis-Golgi marker at this point, as was suggested by the reviewer, and generated the red fluorescent Nb G -tagged cis-Golgi-localizing anchor Man1-RFP-Nb G for the requested control experiments.The endocy c uptake assays with secGFP-SYN and Man1-RFP-Nb G -expressing cells revealed secGFP-SYN signals in the vacuole, but no colocaliza on between the endocytosed secGFP-SYN and the cis-Golgi-localizing Man1-RFP-Nb G was observed, demonstra ng that the endocytosed secGFP-SYN does not reach the cis-Golgi upon endocy c uptake by default and that the pos ransla onal labeling of the cis-Golgi-localizing Nb G -RFP-VSR7 with the secGFP-SYN must have occurred due to the downstream trafficking of the receptor to the TGN/EE.This new result nicely supplements the presented control in which we used Man1-CFP expressing cells in endocy c uptake assays with secGFP-SYN (Fig. 4c) and is therefore inserted as a new panel in the revised Fig 4 as Fig. 4d, with the quan fica on being presented in the new Supplementary Fig. 6.This is also in agreement with the published data showing that the endocy c uptake of such soluble fluorescent proteins leads via the TGN/EE and the MVB/LE to the vacuole, independent of sor ng receptors (Künzl et al., 2016, Nature Plants 2, 16017).However, we were confused regarding the above request to show that "RFP does not go to the TGN."In the context of the trafficking analysis of VSR7 (Fig. 4), RFP is transla onally fused to the Nb Gtagged full-length VSR7, Nb G -RFP-VSR7, and the results show that this molecule does reach/transit the TGN/EE (Fig. 4f).Therefore, we decided to omit this sugges on.Together with the new control, it seems now jus fied to claim that the colocaliza on of the cis-Golgi marker Man1-CFP with the pos ransla onally endocytosed secGFP-SYN-labeled Nb G -RFP-VSR7 (Fig. 4h) demonstrates that Nb G -RFP-VSR7 did transit the TGN/EE, acquired the labeling due to the nanobody-epitope interac on in the TGN/EE and recycled back to the cis-Golgi a erward.

Minor
I think the reference to the numbers of VSRs and RMRs in page 2 refers to Arabidopsis and does not apply to higher plants in general.Response: We thank the reviewer for spo ng this.We have clarified in the manuscript that the numbers given for VSRs and RMRs refer to Arabidopsis.I don't understand the ra onale for choosing the genes authors used for the phylogene c analysis in Suppl 1. Also, because tobacco cells are used in this experiment, it would be be er to show that there are Class III VSRs in tobacco as well.

Response:
We decided to show the phylogene c rela onship of members of the VSR family from different species to emphasize the classifica on of the VSR family.As suggested, we have now also included the three published VSR sequences from Nico ana tabacum and iden fied the VSRs NtVSR1 as class I, NtVSR3 as class II, and NtVSR6 as class III VSRs.For VSR5/6, which also belong to Class III, it has been reported that OX of VSR5 in the vsr1vsr4 double mutant promotes AALP secre on (Lee et al., 2013 h ps://doi.org/10.1104/pp.112.210914).Whether VSR5/6 and VSR7 have different func ons in the same Class III or it is due to dis nct experimental systems should be verified by addi onal experiments, if possible.

Response:
This is an exci ng ques on, but it isn't easy to answer and is even more challenging to inves gate.The func on of sor ng receptors is complex and includes reversible cargo interac on and bidirec onal transport of the receptor.Therefore, it isn't easy to judge "func on" in general.Lee et al. (2013, Plant Physiol. 161, 121-133) present a set of sophis cated and excep onally well-executed experiments.They used vacuolar trafficking-impaired vsr1vsr4 mutant protoplasts for coexpressing increasing amounts of HA-tagged VSRs (either VSR1, VSR4, or VSR5 but not the VSR6 or VSR7) with a constant amount of the soluble vacuolar cargo AALP:GFP, and analyzed the amount of reporter in cell extracts and culture medium by SDS-PAGE/WB to assess transport and to draw conclusions about the involvement of the respec ve VSR in the transport of the cargo into the vacuole.In the case of the VSR5, increased expression levels of the receptor resulted in an increased amount of "non-sorted" vacuolar cargo that reached the culture medium (Lee et al.,2013, Fig. 6).While it is evident that the expressed VSR5 did not recover the mutant phenotype, it is not clear why the increased amount of the receptor gradually enhanced the vsr1vsr4 phenotype.At first sight, an increase in ligand-bindingcompetent receptors is expected to reduce/prevent secretory loss, while an increase in the number of ligand-binding-incompetent receptors is expected not to alter the number of missorted cargo molecules at all.However, the above experiment (Lee et al.,2013, Fig. 6) revealed a VSR5 dosagedependent increase in the cargo sor ng defect.The most plausible way to explain such an enhancement of the sor ng deficiency of the mutant line is that the expression of the VSR5 also interfered with the transport of the sor ng-relevant endogenous receptors, which reduced the already low sor ng efficiency of the mutant even further.One possibility for such influences could be the compe on between the endogenous receptors and the expressed receptors for coat proteins or other transport machinery components, either for the anterograde or retrograde direc on, that could cause the transport to collapse, and receptors would con nue to accumulate.At such condi ons, accumula ng even a ligand-binding competent VSR in a compartment that does not provide ligand-binding condi ons would not reduce the secretory loss of cargo in the mutant.
In an a empt to tackle the ques on regarding similar or different func ons of class III VSRs, we have generated a VSR5-based sensor system and assessed the ligand-binding ability of the VSR5 via FRET/FLIM, similar to what was performed for the analysis of the VSR7 (shown in Fig. 1e).The confiden al Fig. C1 below shows that the ssVSS-carrying ligand Aleu-RFP also triggers the reduc on of the VSR5 sensor's fluorescence life me, while the ctVSS-carrying RFP-AFVY and the nonligand RFP-HDEL does not.This suggests that VSR7 and VSR5 exhibit the same ligand binding competence for the ssVSS-carrying ligand Aleu-RFP in vivo.Regarding the experiment presented by Lee et al., 2013, increased secre on of AALP:GFP by a ssVSSbinding-competent VSR5 in the vsr1vsr4 mutant would argue for a transport compe on situa on between the receptors, as discussed above.Therefore it would be crucial to know the steady-state distribu on of the endogenous VSRs of the vsr1vsr4 line, which are relevant for the AALP:GFP sor ng, and the localiza on of the expressed VSRs regarding their binding/release loca ons.However, VSR7 and VSR5 do not colocalize.Therefore, it must be assumed that their transport mechanisms differ.However, concluding receptor func onality between the class III VSRs VSR5, VSR6, and VSR7 is a very complicated issue that requires complex analysis of their ligand spectra and mapping of their intracellular trafficking routes, which exceeds the scope of this manuscript.At this stage, we can not provide sufficient data for a conclusive answer to this ques on, and we, therefore, prefer omi ng this topic from the manuscript.
Bo om of page 3, RFP-AFVY is not accumulated in the ER, so it should not be surprising that FRET does not occur.

Response:
We are sorry about this mistake.We were surprised by the lack of colocaliza on between the sensor and the ctVSS carrying cargo RFP-AFVY.We have clarified this in the manuscript.

Response:
We thank the reviewer for this informa on, and we have added the no on that the VSR7 has been iden fied in frac ons of immunoisolated SYP61-CFP or vacuolar-type H + -ATPase subunit a1 (VHA-a1)-GFP compartments in proteomic analyses, and we have added the respec ve references Drakakaki et al., 2012, Cell Res. 22, 413-424 andGroen et al., 2014, J. Proteome Res. 13, 763-776, accordingly.There is no data to exclude the possibility that VSR7 is recycled from the MVB, therefore the tle should be an overstatement.

Response:
With all due respect, we do not agree with this view.Even though recycling from the MVB/LE in plants is controversial (Robinson and Neuhaus, 2016, J. Exp. Bot. 67, 4435-4449.), it seems that this case does not apply to the recycling of the VSR7 in par cular.Our newly added quan fica on and sta s cal analysis of the VSR7 trafficking analysis show that the inhibi on of clathrin-mediated trafficking by the expression of the GFP-Hub alters the distribu on of the cis-Golgi-localizing VSR7 and causes its accumula on at the TGN/EE first, while the accumula on at the MVB/LE occurs only a er a prolonged-expression for 48 h.If the demonstrated clathrin-dependent recycling of the VSR7 occurred from the MVB/LE rather than the TGN/EE, the GFP-Hub would have caused the accumula on of the VSR7 at the MVB/LE first, which is not the case.Regarding puta ve recycling of VSRs from the MVB/LE, it is noteworthy to say that -at least to our knowledge -no experimental evidence has been provided that demonstrates that clathrin-coated vesicles transport VSRs with soluble vacuolar cargo to the MVB/LE, or that VSRs depart from an MVB/LE in a clathrin-coated vesicle for retrograde recycling to the cis-Golgi (Robinson and Neuhaus, 2016, J. Exp. Bot. 67, 4435-4449.).It, therefore, seems jus fied to claim that the VSR7 recycles from the TGN/EE to the cis-Golgi.
More careful Discussion would be needed.In the Discussion, the authors state that VSR4 and VSR7 should have the same velocity of anterograde transport in the Golgi.What is the ra onale for this?

Response:
We are sorry that this has caused confusion.In the Discussion, we did not intend to state that "VSR4 and VSR7 should have the same velocity of anterograde transport in the Golgi" in general.We referred to a model situa on in which the anterograde transport of membrane proteins across the Golgi stack would occur via a matura on-based cisternal progression mode that employs the Golgiassociated COPI transport vesicles only for the retrograde recycling of Golgi-resident proteins but not for selec ve anterograde transport of cargo proteins.For this situa on, we assumed that the transport of membrane proteins would occur at the same speed.We have clarified this in the manuscript.

Reviewer #3 (Remarks to the Author):
In this manuscript Shao et al., inves gates the trafficking routes of VSR7, an understudied vacuolar sor ng receptor and suggest that VSR7 bind cargoes in the cis-Golgi to then release them in downstream TGN/EE compartment.In the paper they use a previously published strategy based on the interac on between GFP and a GFP nanobody to test the trafficking of VSR7.The sensor is able to detect VSR7 in the cis-Golgi.They use FLIM to test interac on of different vacuolar cargoes and show that VSR7 is responsible for the trafficking of an Aleu-RFP cargo.They localize the full length receptor to the cis-Golgi and develop an assay to show that VSR7 recycles back to the Golgi from TGN/EE.Addi onally, they show that VSR7 trafficking is clathrin dependent by using a clathrin mutant.In conclusion they suggest intra Golgi transport of a sub-set cargoes in plants may be a receptordependent process.This is a very interes ng model and a novel concept across species as intra-Golgi transport in mammals (debated) and in yeast is thought to be driven by cisternal matura on.In my opinion this is a very bold claim which in this case is unfortunately not supported by the data.

Response:
With all due respect, we do not claim that the intra-Golgi transport of soluble vacuolar proteins across the stack is a receptor-dependent process.However, we agree with the reviewer that our data would not support such a claim.In transporta on, the term "receptor-dependent" refers to a transport situa on that does not occur without the receptor.This does par cularly not apply to the anterograde intra-Golgi transport of soluble proteins across the stack.The Golgi stack is a central part of the secretory pathway that is taken by soluble secretory proteins, which don't possess sor ng signals, by default.Likewise, soluble Golgi-transi ng proteins that carry sor ng signals for post-Golgi loca ons, like the vacuole, are also secreted in mutants that lack the respec ve receptors.Therefore, intra-Golgi transport across the stack can't be termed receptor-dependent.However, post-Golgi transport of soluble proteins to loca ons other than the plasma membrane requires sor ng signals and, consequently, the interac on with sor ng receptors, as is the case for the VSR-mediated sor ng of soluble vacuolar proteins.In this regard, we demonstrate that the VSR7 binds specifically to the ssVSS-carrying cargo Aleu-RFP in vivo.We show that the VSR7 binds Aleu-RFP in the ER, the cis-Golgi, but not in the TGN/EE.Our localiza on analysis shows that the VSR7, in contrast to other VSRs, localizes at steady state condi ons at the cis-Golgi, trafficks to the TGN/EE for the release of ligands, and we provide evidence for its clathrin-dependent retrograde recycling to the cis-Golgi.
It would need careful evalua on and quan fica on of the imaging data before it can be published.I cannot suggest this work and I feel like I am unable to evaluate this work un l proper quan fica on is carried out.The authors must carry out quan fica on and sta s cs on independent experiments.With crops with one single dot structure, it is impossible to evaluate how sound the data is… For example, in Figure 2B I do see colocaliza on between ManI and Aleu which makes me worry that the authors may have biasedly picked crops.

Response:
We agree with the reviewer and apologize for leaving the impression of having biasedly picked crops.The lack of quan fica on of the CLSM localiza on data has also been raised by reviewer #2, and we have replied to this in detail above.In short, we have quan fied the colocaliza on of the respec ve receptors and marker proteins for the analyses of receptor localiza on and trafficking presented in Fig. 3, Fig. 4, and Fig. 5 and Supplementary.Fig. 8 by calcula ng Pearson's correla on coefficient (r P ) and nonlinear Spearman's rank correla on coefficient (r S ) of the respec ve fluorescent signals and performed a sta s cal analysis of the PSCs.We present these data in three new supplementary figures, Supplementary Fig. 5, Supplementary Fig. 6, and Supplementary Fig. 7.We appreciate this sugges on and acknowledge that the added data have significantly strengthened our claims.I understand that the author claim that cargo binds to the receptor in the cis-Golgi based on their FLIM interac on data (of which I am not an expert and cannot evaluate the soundness…).However, I fail to see data in support of release of cargo in the TGN/EE.

Response:
We appreciate this comment.Visualizing the compartment-specific ligand release in vivo is most challenging, par cularly in the TGN/EE, because neither the VSR7 nor the iden fied ligand Aleu-RFPor any other soluble vacuolar protein localizes at the TGN/EE at steady-state condi on.We concluded that the VSR7 releases the ligand in the TGN/EE since it is the first compartment of the vacuolar transport pathway, that, in contrast to the ER, cis-and trans-Golgi, provided no colocaliza on-based evidence of an occurring interac on between the VSR7 sensor and the iden fied ligand Aleu-RFP (Fig. 2a-d).In the protoplasts, the red fluorescent protein-tagged vacuolar cargo causes strong red vacuolar background signals and, addi onally, accumulates in the MVB/LE (red punctae) at steady-state condi ons, while the green fluorescent sensor is localized in TGNs/EEs (green punctae), that in cor cal view appear on top of the strong vacuolar background.In this situa on, selec ng regions of interest for quan fica on is challenging.To solve this problem, we implemented FRET/FLIM to quan fy the VSR7 sensor-ligand interac on in the TGN, which we included as a new panel in Fig. 2, Fig. 2e).In this analysis, we included the drug concanamycin A (Conc A), an established inhibitor of the TGN/EE-localizing vacuolar-type H + -ATPase subunit a1 (VHA-a1) that alters the pH of the TGN and thus blocks the post-TGN/EE transport to the vacuole.The new Figure 2e confirms that the ligand Aleu-RFP does not interact with the VSR7 sensor in the TGN/EE at control condi ons but shows strong interac on in the presence of the drug.This is the first demonstra on of a pH-dependent VSR-ligand interac on in vivo and provides now strong evidence for the TGN/EE as the ligand-release compartment of the VSR7.I also recommend that the authors move away from using non-color blind friendly red and green to show their microscopy images.

Response:
We are aware that this is an important issue, and we envisaged the presenta on of the microscopy data following guidelines for color blindness provided by Wong, 2011, Nat. Methods 8, 441; This, however, did not yield sa sfactory results regarding the recogni on and judgment of the varying combina ons of overlapping signals in the three-channel imaging analysis in the context of the compartment-specific signal pa ern of the protoplasts.
Confiden al Figure C1: CLSM analysis of VSR5 sensor-ligand interac on in tobacco mesophyll protoplasts.(a) FLIM assessment of VSR5 sensor-ligand interac ons, showing I) Energy donor (GFP-CNX) only, or the assembled VSR5 sensor GFP-CNX/LBD5-NbG with II) Aleu-RFP, III) RFP-AFVY, IV) RFP-HDEL or V) the GFP-RFP dual-color sensor GFP-CNX/LBD5-RFP-NbG. Fluorescence life mes are given as GFP-CNX fluorescence life me in ns as box plots.Significance was calculated using oneway ANOVA, followed by Tukey's HSD test (****P <0.0001 compared with every other group; NS, not significant, N=10-15 cells).Images in the rows below the chart show false color fluorescence life me analysis (FLIM), and the respec ve localiza on analysis of the proteins (green channel, red channel, and merge).Scale bars = 5 µm.(b) Control of expression of the VSR5 sensing unit.SDS-PAGE/WB analysis showing the expression of the HA-tagged sensing unit LBD7-NbG in coexpression with the ER anchor GFP-CNX and the respec ve RFP-fusion proteins used for the FLIM analysis shown in a, using an -HA an bodies.